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Tuned vibration modes in a miniature hearing organ:Insights from the bushcricketAnna Vavakoua , Jan Scherberichb , Manuela Nowotnyb , and Marcel van der Heijdena,1

aDepartment of Neuroscience, Erasmus MC, University Medical Center Rotterdam, 3000 CA, Rotterdam, The Netherlands; and bInstitute for Zoology andEvolutionary Research, Friedrich Schiller University Jena, 07743 Jena, Germany

Edited by Daniel Robert, University of Bristol, Bristol, United Kingdom, and accepted by Editorial Board Member Thomas D. Albright August 17, 2021(received for review March 17, 2021)

Bushcrickets (katydids) rely on only 20 to 120 sensory unitslocated in their forelegs to sense sound. Situated in tiny hear-ing organs less than 1 mm long (40× shorter than the humancochlea), they cover a wide frequency range from 1 kHz upto ultrasounds, in tonotopic order. The underlying mechanismsof this miniaturized frequency-place map are unknown. Sensorydendrites in the hearing organ (crista acustica [CA]) are hypoth-esized to stretch, thereby driving mechanostransduction andfrequency tuning. However, this has not been experimentally con-firmed. Using optical coherence tomography (OCT) vibrometry,we measured the relative motion of structures within and adja-cent to the CA of the bushcricket Mecopoda elongata. We founddifferent modes of nanovibration in the CA that have not beenpreviously described. The two tympana and the adjacent septumof the foreleg that enclose the CA were recorded simultaneously,revealing an antiphasic lever motion strikingly reminiscent of ver-tebrate middle ears. Over the entire length of the CA, we wereable to separate and compare vibrations of the top (cap cells) andbase (dorsal wall) of the sensory tissue. The tuning of these twostructures, only 15 to 60 µm (micrometer) apart, differed systemat-ically in sharpness and best frequency, revealing a tuned periodicdeformation of the CA. The relative motion of the two struc-tures, a potential drive of transduction, demonstrated sharpertuning than either of them. The micromechanical complexity indi-cates that the bushcricket ear invokes multiple degrees of free-dom to achieve frequency separation with a limited number ofsensory cells.

insect hearing | optical coherence tomography | crista acustica |Mecopoda elongata

A hearing organ converts sound-induced mechanical vibra-tions to receptor potentials that are further processed along

the auditory pathway. Bushcrickets (katydids) Mecopoda elon-gata (Insecta, Orthoptera, Tettigoniidae) have a miniature hear-ing organ, the crista acustica (CA), that is located in their fronttibia and can sense frequencies up to 70 kHz (1). To achieve that,they rely on less than 50 sensory units (2, 3), each consisting ofa sensory cell, a cap cell (CC), and a scolopale cell (Fig. 1D; seeFig. 3B). The total length of the CA is ∼0.9 mm, which is ∼40times shorter than the uncoiled cochlea in humans. Still, the CAwith its limited cellular machinery is able to mechanically sepa-rate a wide range of frequencies, but the underlying mechanismsare not well understood.

In bushcrickets, the sound-induced nanovibrations reach thesensory tissue through two routes. The main source of airbornesound is the spiracle (4), a horn-shaped opening in the thoraxthat delivers and enhances the sound input to the CA via anacoustic trachea (5–7). Second, the two tympana on the ante-rior and posterior sides of the front legs are also reported todeliver sound to the sensory tissue (8, 9). The sensory units ofthe CA are surrounded by haemolymph fluid and are protrud-ing from the dorsal side of the dorsal wall (DW) of the acoustictrachea. Each ciliated tip of a sensory dendrite is embedded inthe cap of a CC (Fig. 1D; see Fig. 3B). These CCs are part of acontinuous tectorial membrane that connects the sensory units

(10). Transduction takes place as the mechanosensitive channelson the sensory dendrite open due to displacement of the CC(11, 12). The CA in bushcricket ears is tonotopically organizedalong the proximal–distal axis (12–14) following an anatomicalgradient, with increasing height and width of the sensory units(3, 15) toward the proximal end. Mechanical measurements ofsound-induced responses revealed that high frequencies inducegreater motion magnitude in the distal part of the CA (16, 17).Spatial phase gradients in tone-evoked vibrations indicate theexistence of waves traveling from the distal, high-frequency endto the proximal end of the CA (16). Whereas traveling waves inthe mammalian cochlea accumulate several cycles of phase delay(18), the total phase accumulation in the bushcricket CA is only0.5 to 1 cycle (19). The exact mechanisms that drive transductionand tonotopy in the CA are undefined.

Recently, it was reported that the mechanical responses of theCA show different frequency tuning than the neural responseat the same location (12). The CCs are located dorsally to theDW, and the dendrite of the sensory neuron is approximatelyperpendicular to the DW. The relative motion between the twostructures may drive the opening of the transduction channelsand could explain discrepancies between mechanical and neuraltuning. However, it has not been possible to probe such rela-tive motion experimentally due to technical limitations of thetechnique used in laser Doppler vibrometry (LDV), which has

Significance

Most hearing organs contain an array of sensory cells thatact as miniature microphones, each tuned to its own fre-quency like piano strings. Acoustically communicating insectslike bushcrickets have evolved miniscule hearing organs, typ-ically smaller than 1 mm, in their forelegs. It is still unknownhow the sensory structures inside the leg vibrate in responseto sound. Using advanced imaging techniques, we metic-ulously mapped the nanovibrations in the bushcricket ear.We discovered a complex motion pattern in which structuresseparated by only 1/50 mm showed systematic tuning differ-ences. Despite the insect ear’s tiny dimensions, its mode ofoperation strikingly resembled that of vertebrate ears. Appar-ently, evolution has provided similar solutions to the spectralprocessing of sounds.

Author contributions: A.V., J.S., M.N., and M.v.d.H. designed research; A.V., J.S., M.N.,and M.v.d.H. performed research; A.V. and M.v.d.H. analyzed data; and A.V., J.S., M.N.,and M.v.d.H. wrote the paper.y

The authors declare no competing interest.y

This article is a PNAS Direct Submission. D.R. is a guest editor invited by the EditorialBoard.y

This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).y

See online for related content such as Commentaries.y1 To whom correspondence may be addressed. Email: marcel.vdh.work@gmail.com.y

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2105234118/-/DCSupplemental.y

Published September 22, 2021.

PNAS 2021 Vol. 118 No. 39 e2105234118 https://doi.org/10.1073/pnas.2105234118 | 1 of 10

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Fig. 1. OCT imaging. (A) Illustration of the experimental setup. The fixed animal was placed under the OCT vibrometer, and sound was delivered using anopen-field stimulation paradigm. (B) Schematic drawing of the foreleg that holds the hearing organ, CA, including a proximal–distal/dorsal–ventral sectionof the CA. (C) A cross-section OCT image (“B scan”) obtained from the top view of the hearing organ, following an image section plane as shown in B,across the proximal–distal axis. (D) Sketch relating the B scan to the anatomy across the proximal–distal axis. The DW and the CCs can be identified. The CAposition at the distal end indicated with the green circle is the reference position used in this study to normalize sound-induced vibrations. By convention,all the distances are expressed relative to the red line that illustrates the very distal part of the CA. di, distal; do, dorsal; p, proximal.

been extensively used to measure vibrations in insect hearingorgans (16).

LDV measurements do not provide inherent depth resolu-tion, and the optical inhomogeneities of biological tissues causediffraction, scattering, and lensing effects that adversely affectoptical focus. Therefore, most LDV measurements in the insecthearing organs were done on the most accessible and reflectivestructures, like the tympana (20), antenna (21), and cuticle, asan indirect measurement of organ motion (17). Measurementsof hearing organ motion were also obtained through a trans-parent cuticle (22) or after the surgical removal of the cuticle(16). Optical coherence tomography (OCT) provides opportuni-ties to reliably attribute vibrations to anatomical structures, asit resolves depth and allows for fast nanovibrometry measure-ments (23). OCT has yielded vibration maps of cross-sections ofthe mammalian organ of Corti (24) and the description of differ-ent vibration modes of the cochlear partition (25). The new OCTresults revealed structural interconnections in the micromechan-ics that stirred up the understanding of cochlear function (26) buthave not yet been used to shed light on the operation of insecthearing organs.

In the current study, we used OCT vibrometry to performnoninvasive measurements of nanovibrations in the bushcrickethearing organ. First, we used this technique to describe the rel-ative motion of the two tympana and the septum of the trachea(Fig. 2) in response to broadband, multitone sound stimuli. Next,we approached the CA from the dorsal side (Fig. 1B) to performsimultaneous vibration measurements of the CC and the DW,and this allowed us to determine the relative motion betweenthese closely neighboring structures. We observed systematic dif-ferences in the frequency-place map and sharpness between CCand DW. The relative motion between CC and DW was found tobe more sharply tuned than either of the individual structures.

ResultsThe Relative Motion of the Two Tympana. The two tympana areeasily accessible for imaging in a noninvasive manner, so theywere the first structures we aimed to study. For this purpose, theOCT recording beam was aimed perpendicular to the anteriortympanum. When imaging through the anterior tympanum, therecording beam traverses through the tracheal septum before itreaches the posterior tympanum. Light intensity can be a limitingfactor when imaging with the OCT as highly reflective surfacestend to obscure deeper structures. Our results demonstrate that

with our setup, it is possible to image both tympana and identifythem with certainty (Fig. 2 A and D), which afforded a meansto record sound-induced vibrations on the two tympana (Fig. 2B and C) simultaneously. By doing so, we exclude any effectsthat rotating the preparation has on the acoustic delivery of thestimulus.

The color map in Fig. 2B illustrates how the phase of the vibra-tions is distributed along the B scan (details are in ResponseAnalysis) for a 14.3-kHz stimulus at 79 dB sound pressure level(SPL). The motion of each tympanum had a constant phase pro-file along the recorded width, from the dorsal end until halfwayto the ventral end. The two tympana moved with an ∼0.5-cycle(=180◦) phase difference, and this phase opposition was con-sistent across the whole recorded width of the tympana. Acrossdifferent frequencies (0.4 to 38 kHz), the two tympana sharedsimilar vibration magnitudes (Fig. 2F) and showed an ∼0.5-cyclephase difference (Fig. 2 B and E), confirming previous findings(27). For both tympana, the magnitude followed a dorsal–ventraldisplacement gradient, with the higher values toward the ventralside (Fig. 2C). Both tympana describe a hinged motion anchoredat their dorsal ends (Fig. 2G). These observations were system-atic across all our tympanum measurements (n = 11 bushcricketlegs) (SI Appendix, Fig. S1).

In addition to the two tympana, we were also able to observethe phase of the vibrations on the anterior part of the DW andthe tracheal septum, a structure splitting the acoustic tracheain two compartments, for which no vibration data have beenreported before (Fig. 2B). In Fig. 2B, the anterior end of the CAis seen to move in phase with the anterior tympanum (deepestcyan squares), while the septum moves in phase with the poste-rior tympanum (dark orange squares halfway between the twotympana). These structures are very close to the sensory unit,and their antiphasic motion could be of functional significanceif it resulted in a mechanical stress on the CA that could be feltby the sensory dendrites. We did, however, not further pursuethis analysis here because the anterior–posterior viewing angleis suboptimal for studying relative motions within the sensoryunit; the DW in particular is hardly visible. Instead, we studiedthe DW motion along the dorsal–ventral axis, where it is clearlyvisible.

Micromechanics: Dissecting the Motion within the CA. Aiming toexplore further motions within the sensory units, we imaged theCA through a dorsal view as illustrated in Figs. 1 and 3. That

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Fig. 2. Mechanical responses of the anterior and posterior tympana to sound stimulation. (A) OCT B scan of a front leg viewed through the anteriortympanum. (B) The same B scan overlaid with color-coded phase information for a stimulus frequency of 14.3 kHz presented at 79 dB SPL. Notice that aphase difference of 0.5 cycles is 180◦ (an out-of-phase motion). The hemolymph fluid behind the anterior tympanum caused the position of the dorsalpart of the posterior tympanum to shift to a more posterior position. This optical distortion does not affect the vibration data. The white stripes in thecolor map mark the average phases of the two tympana. (C) The same B scan overlaid with the companion color-coded vibration magnitude informa-tion. (D) Anatomical sketch of a cross-section of the bushcricket front leg. Adapted from ref. 3. (E) The displacement phase difference between posteriorand anterior tympana across different stimulus frequencies. The mechanical measurements were obtained at the positions indicated by the markers inB. (F) Displacement magnitude differences between posterior and anterior tympanum across different stimulus frequencies at the marked positions inB. (G) Two extreme configurations of the tympana within one cycle of stimulus frequency are illustrated with red and blue. Displacement amplitudeis enlarged 5,000× for illustration purposes. The two tympana bulge in (red) and out (blue) synchronously. The septum follows the posterior tympa-num motion direction. The arrows illustrate the magnitude of vibrations, which increases toward the ventral side of the tympanum. The ventral part ofthe tympanum moves ∼3.6 times more than close to the CA at the stimulus frequency. The tympana motion resembles a hinge, anchored at the dor-sal end. More data are in SI Appendix, Fig. S1. a, anterior; do, dorsal; atym, anterior tympanum; hl, hemolymph; ptym, posterior tympanum; s, septum;tr, trachea.

way, our recording beam travels through the CA aligned withthe dorsal–ventral anatomical axis, allowing us to distinguishthese structures in our B scans. As a consequence, motion of theCC and the DW can be disentangled. The mechanical measure-ments were normalized to vibrations recorded in the distal endof the CA. The results presented in detail below come from apreparation where vibrations on the CA were measured throughthe intact dorsal cuticle (BC0007) (details are in Materials andMethods).

Using OCT vibrometry, we can expand the datasets in theexisting literature. Previous studies have reported a tonotopicresponse of the CA with high and low frequencies exciting thedistal and proximal parts of the organ, respectively (13, 16, 17).The tonotopic gradient coincides with a gradient in the size ofthe anatomical structures (3). In Fig. 3, the recordings of the DW(black curves) are shown alongside those of the CC (blue curves).Spatial and spectral patterns of the sound-induced CA motiondemonstrate the tonotopy in two complementary ways. Fig. 3 D–F shows the CA vibration magnitude evoked by three differentfrequency components (6.3, 12.5, and 23.4 kHz) as a function of

longitudinal position, as measured on the DW and the CC. Each ofthese excitation patterns is peaked; with decreasing frequency, thepeak location shifts toward the proximal end. The correspondingphase data (Fig. 3 G–I) display an accumulation of ∼0.5 to 1 cycleof phase from the distal end to the peak region. These phase datayielded estimates of wavelength and phase velocity in the peakregion as detailed in Fig. 3. Wavelength ranged from 345 to 599µm (DW) and from 222 to 370 µm (CC); phase velocity rangedfrom 3.7 to 8.1 m/s (DW) and from 2.4 to 5.2 m/s (CC). Thesevalues are consistent with previous reports in M. elongata (16, 19).

The spectral representation of tuning is illustrated in Fig. 3 J–O by plotting magnitude (Fig. 3 J–L) and phase (Fig. 3 M–O)as a function of frequency recorded at three positions along thetonotopic axis. The tonotopy and phase patterns in Fig. 3 areconsistent with previous reports of CA vibrations (12, 19). Theoverall tuning and tonotopy of the DW are similar to those ofthe CC, but we also found systematic differences between thetuning of the two structures in the 5- to 25-kHz frequency range.The differences in tuning between DW and CC will be detailedbelow.

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Fig. 3. Differences in the tonotopic response and frequency tuning of the DW and the CCs. (A) An anterior–posterior cross-section of the front leg. (B)OCT B scan obtained from the top view of the CA, following an image section plane as shown in A, across the anterior–posterior axis. (C) Sketch relatingthe B scan to the anatomy across the anterior–posterior axis. The height of the CA is in the range of 15 to 60 µm, following the tonotopic axis from distalto proximal (3). d, sensory dendrite; sc, scolopale cell. (D–F) Normalized magnitude of the DW (black lines) and the CC (blue lines) tonotopic response fordifferent frequencies along the CA. Since a single reference was used for both structures (Materials and Methods), the graph conveys their relative vibrationmagnitude. In the axes labels, re ref means relative to the reference. (G–I) Normalized phase of the DW and the CC tonotopic response for differentfrequencies at the same stimulus frequencies as in D–F. DW wavelength estimates: 590, 455, and 346 µm, respectively. CC wavelength estimates: 370, 256,and 222 µm, respectively. These correspond to phase velocities of 3.7, 5.7, and 8.1 m/s (DW), respectively, and 2.4, 3.2, and 5.2 m/s (CC), respectively. (J–L)Mechanical tuning of the normalized magnitude of the DW and the CCs at different positions that are marked by their distance to the distal end of the CA.(M–O) Normalized phase of the DW and the CCs frequency tuning at the same positions as shown in J–L. DW group delay estimates: 344, 294, and 330 µs,respectively. CC group delay estimates: 454, 578, and 578 µs, respectively. (Data are from experiment BC0007, recorded through the intact cuticle. Stimuluslevel is 75 dB SPL.) Population data are in SI Appendix, Fig. S2. a, anterior; p, posterior; do, dorsal; di, distal.

Distinct Modes of Vibration within the CA. When comparing thetonotopic response with three distinct stimulus frequencies, theDW has a wider peak than the CC, and this widening is unilat-eral. Specifically, it is the proximal flank that is different betweenDW and CC (Fig. 3 D–F). The phase data (Fig. 3 G–I) revealthat the DW accumulates less phase between the distal end andthe peak region. The frequency tuning of three different posi-tions along the CA length (Fig. 3 J–L) shows that the DW isless sharply tuned. When recorded at the same distance fromthe distal end, the DW is tuned to higher frequencies than theCC. Finally, the DW phase curves are shallower than their CCcounterparts, implying that DW vibrations have a shorter groupdelay than CC vibrations. These systematic differences betweenCC and DW vibration were observed in all animals tested (SIAppendix, Fig. S2). In the curves of Fig. 3 M–O, group delayfor the CC exceeded that for the DW by 110, 284, and 248 µs,respectively. Since group delay is a metric of the latency withwhich the vibration energy arrives at the recording site (28),this suggests that the acoustic energy reaches the DW prior toreaching the CC.

Two Parallel but Shifted Place-Frequency Maps for the DW and theCC. Are the tuning differences between the two structures appar-

ent on the whole length of the sensory tissue? To test for that, wemeasured vibrations on the DW and the CC along 800 µm ofthe CA using 33-µm steps, while presenting stimulus frequenciesfrom 0.4 to 50 kHz in ∼950-Hz steps. The spatial and frequencysampling of our measurements allowed us to construct heat mapsof the tuning of the two structures (Fig. 4 A–D). This way ofpresenting magnitude (Fig. 4 A and B) and phase data (Fig. 4C and D) combines the spatial and spectral tuning shown sepa-rately in Fig. 3 D–O. We were able to determine the location ofthe spatial peak and the spatial 5-dB down points for each stim-ulus frequency, as indicated in the graphs. Consistent with theindividual spectral and spatial tuning data in Fig. 3, the DW hada wider peak width than the CC over the entire frequency range,and the DW peaked more proximal than the CC peak. The phaseresponses confirm the greater accumulation of phase in the CCcompared with the DW.

A detailed quantitative analysis of the tuning differencesbetween CC and DW reveals that the tuning of the DW is shiftedproximally compared with the CC (Fig. 4 E and F). The tuningof the DW is wider than that of the CC (Fig. 4G). The systematicdifferences between the tuning of the CC and the DW span theentire length of the hearing organ, as well as the entire spectralrange tested (2.4 to 50 kHz). When averaged over this range, the

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Fig. 4. Longitudinal vibration maps reveal tuning differences between the DW and the CC. (A and B) Normalized magnitude responses of (A) the DWand (B) the CC across different frequencies and recorded positions along the CA. The spatial peak for each frequency (black dot) and the associated 5-dBbandwidth (black lines) are overlaid. (C and D) Normalized phase responses of (C) the DW and (D) the CC across different frequencies and recorded positionsalong the CA. The spatial peak for each frequency (black dot) and the associated 5-dB bandwidth (black lines) are overlaid. (E) The frequency-dependentspatial peak per stimulus frequency for the DW and the CC. (F) The frequency-dependent spatial peak per stimulus frequency for the DW and the CC. Inthe log-lin axis, both tonotopic maps are approximately straight and parallel but shifted relative to one another. (G) The −5-dB peak width vs. stimulusfrequency for the DW and the CC. (Data in A–G come from experiment BC0007. Stimulus level is 75 dB SPL. More data are in SI Appendix, Fig. S3.) (H) Theplace-frequency map of the DW across n = 10 hearing organs. (I) The place-frequency map of the CC across n = 10 hearing organs. (J) Median slopes of the10 place-frequency maps are not significantly different. Data are extracted from H and I. (K) Population data of the relative peak position of CC vs. DWwith N = 10 legs. Spatial peak locations were pairwise subtracted between DW and CC for each frequency. The difference was statistically evaluated using2-kHz-wide bins. The boxes have lines at the lower quartile, the median, and the upper quartile values. Whisker pairs at each box show the extent of therest of the data, with outliers marked by red plus signs. Individual bin sizes n are indicated. Over most of the frequency range, spatial peaks in the CC wereshifted distally compared with the DW. (L) Corresponding comparison of the spatial peak width between DW and CC; the same N = 10 hearing organs asin K. Denoting the 5-dB down peak widths of the DW and the CC by WDW and WCC , respectively, their normalized difference is ∆ = (WCC −WDW )/WDW .Statistical analysis and layout are the same as in K. The spatial peaks in the CC were systematically narrower than the peaks in the DW. re ref, relative to thereference.

tuning difference amounts to 0.19 octaves in spectral terms (Fig.4E) and 31 µm in spatial terms (Fig. 4F).

The current dataset allows for population metrics of the tun-ing of the two structures. The place-frequency maps of the DWand the CC of 10 hearing organs are shown in Fig. 4 H andI, respectively. Place-frequency maps of the DW and the CCmotion from 10 different CAs show overlapping results, whichimply that the used method of anchoring the different mea-surement positions to the distal end of the CA is robust. TheDW and the CC share a similar slope on their place-frequencymaps. Each octave of “best frequency” occupies 170 µm alongthe CA for both the DW and the CC (Fig. 4J), a stretch contain-

ing approximately nine sensory cells. The systematic differencesin tuning between DW and CC were observed in all 10 animalstested, both in terms of peak location (Fig. 4K) and peak width(Fig. 4L).

Internal Motion within the CA. The relative motion of the DW andthe CC is a candidate for the “drive” of transduction, as thetwo structures coincide with the two ends of the sensory den-drite, which is oriented perpendicular to the DW (Fig. 3C andSI Appendix, Fig. S5). The motion here is measured along thedorsal–ventral axis that is approximately parallel to that of thedendrite tip. The CC/DW magnitude ratio (Fig. 5A) shows that

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Fig. 5. Relative motion of CCs and the DW. (A) Color-coded magnitude difference (in decibels) between the CC and the DW mechanical response. Markers(point = DW, circle = CC) illustrate the spatial peak for each stimulus frequency, as in Fig. 4E. (B) Phase difference between the CC and the DW mechanicalresponse of the same data as shown in A. (C) Magnitude of the vector difference between the DW and the CC mechanical response for the same data asdisplayed in A and B. (D–F) The tonotopic response of the DW, the CC, and their vector difference shown for three stimulus frequencies as indicated (datain A–F come from experiment BC0007). Stimulus level is 75 dB SPL. More data are in SI Appendix, Fig. S2. re ref, relative to the reference.

the two structures share similar magnitude of vibrations in posi-tions distal to the spatial peak for each frequency. Proximal tothe spatial peak, the DW magnitude dominates, resulting in neg-ative dB values for the ratio. This analysis further characterizeshow the CC magnitude decreases more distally than the DWmagnitude (compare with Fig. 4).

The direct comparison of the phase between CC and DW (Fig.5B) reveals that the two structures move in phase at positionsdistal to the spatial peak. Consistent with the examples in Fig.3, the phase difference increases drastically at locations proximalto the spatial peak of each frequency. For most frequencies, theDW accumulates a phase lead over the CC of ∼0.25 cycles overa distance of just 30 to 70 µm proximal to the spatial peak, butfor some frequencies, the difference is larger (up to ∼0.5 cycles).The location where the phase difference occurs coincides withthe divergence of the amplitudes, again consistent with the threefrequency examples shown in Fig. 3 D–I.

We further assessed the relative motion of CC and DW bycomputing the vector difference of their mechanical motions(i.e., taking into account both their relative amplitude and phase)(Fig. 5C). The spatial tuning of the vector difference was some-what sharper than that of either structure (e.g., the 5-dB downpeak widths in Fig. 5D were 286, 202, and 187 µm for the DW,CC, and vector differences, respectively). Toward the distal end,its magnitude was smaller than either individual motion. Thisis further illustrated in Fig. 5 D–F, which shows the tonotopicresponse of the DW, the CC, and their relative motion at threedifferent stimulus frequencies: 9.4, 18.4, and 30 kHz. The dif-ferences are asymmetric; it is primarily the distal flank of thetonotopic response that is steeper for the vector difference datathan it is for the DW and the CC.

The spectral data (magnitude vs. frequency) reveal a consis-tent asymmetric sharpening of the tuning of the vector differ-ence data compared with the DW and the CC. It is primarilythe steeper low-frequency flank that distinguishes the vectordifference data from the data of DW and CC alone. The spec-tral tuning of the vector difference is shown for a total ofnine experiments in SI Appendix, Fig. S2; a further example isshown in Fig. 6E.

DiscussionIn summary, we recorded sound-induced vibrations in thebushcricket hearing organ using OCT vibrometry. The depthresolution of this technique yielded an unprecedented set ofmeasurements. First, we measured the sound-induced motion ofthe anterior tympanum and the posterior tympanum at the sametime. Our measurements confirmed that the two tympana bulgedin and out simultaneously (27, 29) (Fig. 2E). Second, the septumbetween them followed the motion of the posterior tympanum(Fig. 2B). Third, we made a detailed and quantitative compar-ison between the sound-induced vibrations of the DW and theCC. The responses of both structures were tuned (Fig. 3), andthere exist systematic differences between them in their tuningacross the whole length of the sensory organ (Fig. 4). The dif-ferential motion of these structures was more sharply tuned thanthe absolute motion of either of them (Fig. 5 and SI Appendix,Fig. S2).

The tympana of the bushcricket hearing organ have beenstudied extensively, but their contribution to the hearing func-tion is not entirely clear. Previous studies (17, 27) reported thatthe anterior and posterior tympana in different Tettigoniidaespecies moved inward and outward synchronously in responseto sound. With LDV, one cannot measure the tympana simulta-neously, and rearranging the preparation across recordings mayhave affected the microacoustic environment in these studies.Our simultaneous recordings provide a direct test of the relativemotion of the tympani. Our data confirm and extend findings ofprevious studies (17, 27). Not only the two tympana, but also theadjacent septum bulge in and out simultaneously. This antipha-sic motion may periodically stretch and squeeze the DW andthe dorsally placed sensory epithelium. The dorsal part of thetympanum sits on top of incompressible hemolymph, so acousticstimulation may cause a translocation of the CA. Such a cou-pling somewhat resembles the scheme proposed by ref. 17 inwhich the dorsal part of the tympana compresses the hemolymphoverlying the CC, but the latter scheme depends on a tympanalplate mechanism in ref. 17 that we did not find in M. elongata.The resemblance of the two schemes is that CA motion is notdirectly driven by the air pressure in the trachea but indirectly,

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A B C D

E

The DW and CC move in

phase, with similar magnitude

along the dorsal-ventral axis.

The magnitude of their vector

The magnitude of the CC

motion is smaller than the DW.

between the motion of the two

structures will further increase

the magnitude of their vector

The CC and the DW move with

similar magnitudes. The CC

motion direction is not aligned

with the recording beam,

resulting to smaller recorded

magnitude.

The CC and the DW both

have complicated tilted

elliptical motions.

BC0017L

dendrite

cap cell

scolopale cell

dorsal wall

recording beam

f < 8 kHz f > 8 kHz

1 2 5 10 20 50−20

−10

0

10

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30

40

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nitu

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B re

ref)

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Fig. 6. Different geometric interpretations of the relative motion between DW and CC observed in this study. (A) In the absence of relative motion(here, <8 kHz; light green background), the DW and the CC move in phase and with similar magnitude along the dorsal–ventral axis (aligned with ourrecording beam). (B–D) Potential internal motion patterns that may underlie the observed differences in magnitude and phase (here, >8 kHz; light purplebackground). (E) Tuning curves of DW, CC, and their vector difference recorded at the medial part of the CA (experiment BC0017L, 79 dB SPL; BF, bestfrequency). Population data are from SI Appendix, Fig. S2.

either by the anterior tympanum and the septum through its con-nection to the posterior tympanum (Fig. 2) or via the tympanalplate and the hemolymph (17). The antiphasic motion of thetympana is likely to cause (or reflect) air pressure variations inthe trachea, which will push and pull the adjacent DW, but theair–hemolymph interface may render this direct route inefficient.Intriguingly, the double-hinged motion (Fig. 2) shows a strikingresemblance with the coupling of sound into vertebrate innerears, where a lever system (middle ear ossicles) converts thelarger motion of a larger surface (eardrum) to smaller motionsof a smaller area (stapes). In vertebrate ears, this configurationprovides an impedance match between airborne sound and thevibrations in the fluid-filled inner ear. The hinged motion of thebushcricket tympana and adjacent septum may well serve thesame purpose.

Bangert et al. (27) reported that blocking the trachea spira-cle with Vaseline decoupled the two tympana. Repeating theseexperiments while using OCT will allow the simultaneous mea-

surement of the two tympana, the tracheal septum, and theCA. This may help to further disentangle the contributionof different acoustics paths to the bushcricket hearing organ.Phase differences along the ventral–dorsal axis of the tympa-num, as shown in other bushcricket species (17), were not presentin M. elongata.

The mechanotransduction channels in the CA sensory neuronsare probably located at the tips of their cilia (11, 12), and they arecommonly assumed to open and close upon longitudinal stretch-ing of the sensory dendrite’s cilium (30). Hummel et al. (12) in2016 observed additional microtubules at the level of the scolo-pidial cap. They surrounded the ciliary tip and could thereforeanchor filaments relevant to gating (31). In that case, transduc-tion is likely to be driven by any relative motion between the DWand the CC since this would stretch and shorten the dendrite. Fig.6 illustrates several potential realizations of this relative motion.A relative motion between the two structures along the dorsal–ventral axis would elongate and shorten the dendrite (Fig. 6B).

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An anterior–posterior motion of the CC could also drive trans-duction, by pivoting of the sensory dendrite (Fig. 6C). Finally,a more complex elliptical motion of the two structures is possi-ble, which could result in a frequency-dependent stretching andpivoting of the sensory dendrite (Fig. 6D). As will be discussedfurther in the following paragraphs, more measurement anglesare needed to disentangle the different components of motionand thus, find the correct interpretation of the internal motion.

Our simultaneous recordings of the DW and the CC are afirst step toward answering these questions. The systematic dif-ferences in tuning between the DW and the CC (Figs. 3 and5 and SI Appendix, Fig. S2) indeed reveal a nontrivial relativemotion between different structures within the CA. Stated differ-ently, acoustic stimuli evoke a periodic deformation of the CA.Importantly, this micromechanical motion is sharply tuned. Iftransduction is indeed driven by such micromechanical motion,our data may explain discrepancies in best frequency and tun-ing sharpness between mechanical and neural tuning in thebushcricket CA (12). In particular, it would explain the sharpertuning of the neural response compared with the mechanics. Apromising way to address such questions in future studies is tocombine OCT vibrometry with neural recordings in the samehearing organ.

Our observations have implications for the understandingof the physical principles shaping the CA vibrations. Despitetheir close vicinity (15 to 60 µm apart, depending on longitu-dinal position), the DW and the CC differ in response phase,best frequency, and bandwidth. This calls for an explanationin terms of vibration mechanics. It is possible that the lon-gitudinal stiffness and size gradients of the DW provide apreliminary stage of frequency selectivity, which is then fur-ther sharpened by local resonances of the individual scolopedia.The larger group delay of the CC compared with the DW(Fig. 3) is consistent with such a two-stage filtering. The hypo-thetical scheme is also reminiscent of the mammalian cochlea,where a physiologically robust, weakly tuned response is comple-mented by a physiologically vulnerable, sharply tuned response(18). Further exploration of such hypotheses will require amore detailed characterization of the micromechanical motionof the CA using OCT, combined with mathematical or numericalmodeling.

The mapping of a wide audio range onto a short (<1-mm)sensory organ necessarily leads to a very dense representation offrequencies. The slope of the frequency-place map amounted to∼170 µm per octave and was nearly identical in DW and CC (Fig.4J). In comparison, the length of the unrolled cochlea is 35 mmin humans and 11 mm in gerbils, and the tonotopic slope in thesespecies is 5,000 (32) and 1,500 µm per octave (33), respectively.Despite these large quantitative differences, it is remarkable thatthe tonotopic map is well represented by a straight line on a log-frequency scale in both the bushcricket (Fig. 4 H and I) andmost mammals [with deviations occurring only at very low fre-quencies <500 Hz (34) and in some echolocating species thathave an acoustic fovea (35)]. This striking similarity suggeststhat, despite vast anatomical differences (e.g., the basilar mem-brane is an interface between two fluids, whereas the DW is aninterface between air and fluid) (SI Appendix, Fig. S5), commonmechanisms may underlie frequency selectivity in the hearingorgans of insects and mammals. Since these mechanisms areequally poorly understood in mammals and insects, any progressin the field of insect hearing may advance our understanding ofmammalian hearing and vice versa. Species with well-accessiblehearing organs like the bushcricket are highly suited for suchcomparative studies.

In mammals, the tonotopic map directly determines the den-sity of neural representation of frequency bands because thespacing of sensory cells is uniform over the cochlear length. Thus,a typical (nonfoveal) mammalian map reveals an equal number

of neural frequency channels per octave over most of the range,with a somewhat poorer representation toward the very lowestfrequencies. In M. elongata, the lower frequencies are also lessdensely represented but this time because of the wider spacingof proximal sensory units (3). It is unclear whether this similarityis rooted in a common functional requirement.

In the context of comparative hearing science, it is also note-worthy that the spatial phase patterns in bushcrickets (e.g.,Fig. 3) have their main features in common with those in themammalian cochlea: an initial flat portion ending in a sharpdownward kink followed by a steep downward slope endingin a plateau (36). Phase accumulation has been interpreted interms of traveling waves in both mammals (32) and bushcrick-ets (19). The amount of phase lag accumulated at the peaklocation is larger in mammals (∼1.5 cycles) (18) than in thebushcricket (∼0.5 cycles), and this may reflect sharper tuningin the mammals (37). With all the similarities, one marked dif-ference between the bushcricket and mammalian cochlea is theeffect of sound intensity. Cochlear responses are very nonlin-ear; they show strongly compressive growth over a large dynamicrange [68 dB of compression over a 96-dB range (18)]. Changesin sound intensity strongly and systematically affect sharpness ofcochlear tuning and peak location of the wave (18) as well as itswavelength, phase velocity, and group velocity (28, 36). In com-parison, the bushcricket CA is much more linear, only showinga slight compression (7 dB of compression over an 80-dB range,mostly concentrated at intensities >100 dB SPL), and there isno apparent effect of intensity on sharpness of tuning or wavepropagation (16).

Although OCT vibrometry is a powerful technique for thestudy of hearing organs, it is important to briefly consider somelimitations and potential ways to overcome them. Recordedvibrations are projections of the actual motion onto the record-ing beam. The more aligned the two are, the closer the measuredmagnitude resembles the actual magnitude. For our DW andCC recordings, we aligned the recording beam with the dorsal–ventral axis, which is perpendicular to the anterior–posterioralignment used for our tympanum recordings. A direct compar-ison between tympanum and CA motion magnitude is thus oflimited scope. Our alignment choices optimize consistency withexisting literature, but it is well possible that there exist otherfunctionally relevant directions of motion between the DW andthe CC that we failed to measure (Fig. 6). For a discussion ofthe spatial and directional interpretation of OCT vibrometry, werefer to ref. 25.

These geometric unknowns caution against oversimplification.It may appear attractive to interpret the different spatial phaseprofiles in CC and DW in terms of two “parallel waves” trav-eling at different speeds, but they may as well reflect a gradualchange in vibration mode of a single wave (38). A more com-prehensive exploration of the micromechanics of the bushcricketCA will require measuring the same structures from differentangles, so that different directions of motion can be disentangled.In the current study, we did image the CA from both an anterior–posterior view and a dorsal–ventral view. At this stage, however,we cannot determine the direction of motion with certainty. Thiswould require a reliable method of registration, ensuring thatthe exact same location is targeted across viewing angles. Inthe literature, the lateralization of the CC responses toward theanterior side of the CA has been described (39). With a vali-dated registration method, one could record the CC from thetwo angles mentioned above, test whether the CC indeed pivotsaround the scolopale cell, and assess the relevance of this motionfor tuning.

Other complications may arise from uncertainties in interpret-ing OCT images in heterogeneous tissues. Our study demon-strates that OCT produces anatomically faithful images of thebushcricket hearing organ (Figs. 1 B–D, 2 A–C, and 3B). The

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same technique has been used to study the morphogenesis ofinsects in a noninvasive manner (40) and insect heart pulsations(41). OCT imaging is based on the delay times of backscat-tered light from different depths of the sample, which not onlydepend on the path length but also, on the speed of lightof the medium as quantified by its refractive index n . For ahomogeneous medium, the delay times are easily correctedfor the refractive index. The insect leg, however, is hetero-geneous; it is partly filled with fluid (hemolymph, n ≈ 1.5)and partly with air (n ≈ 1). Fig. 2 illustrates this; the dorsal,hemolymph-filled part of the leg is distorted compared withthe ventral, air-filled part. Detailed knowledge of the refractiveindex of the imaged structures would enable one to correct forsuch imaging artifacts, but the optical characterization of thebushcricket hearing organ exceeded the purposes of the currentstudy.

This study has employed the opportunities for studyingnonvertebrate hearing organs offered by OCT vibrometry. Itrevealed complexities in the micromechanics of the bushcricketCA that had not been found with previous methods. On the onehand, these findings bring new challenges in understanding insecthearing. On the other, they offer new views and suggest newsolutions to known fundamental problems, including the deliv-ery path of acoustic energy to the sensory tissue, the biophysicsof auditory frequency selectivity, and the exact mechanisms thatdrive transduction. From a wider perspective, current technol-ogy facilitates the comparative study of hearing organs in generaland the similarities and differences between hearing in insectsand mammals in particular. For all these scientific problems,we now have the technical means to explore them. This opensup a wide range of theoretical and experimental exploration ofhearing in miniaturized systems, with numerous technical andscientific applications.

Materials and MethodsAnimal Preparation. In total, 19 bushcrickets of the species M. elongatawere used for in vivo sound-induced vibration measurements on the CAand the tympana. CA recordings were made on 11 individual tibial hear-ing organs belonging to nine animals. The animals were anesthetized bylowering their body temperature after placing them in a container filledwith ice. When they were not responsive (usually after ∼10 min) and theirmandibles were loose, their middle and rear legs were removed. The ani-mal was placed with its thorax on the animal holder, and it was stabilizedusing melted medical wax. The front legs were stabilized in a positionwhere the CAs were accessible to the OCT beam. In two particular exper-iments (BC0007, BC00013), it was possible to record through the intactcuticle. The individual measurements that are shown in the current reportcome from experiment BC0007. For other animals, the dorsal cuticleobscured the view of the CA. For that reason, it was removed surgi-cally and replaced by a glass coverslip. After the cuticle removal, the CAswere inspected using OCT imaging to ensure they were not harmed bythe preparation. Viewing through the glass coverslip allowed us to iden-tify the underlying structures and perform mechanical measurements, andit is a common method in mammal hearing research (42). As was thecase in previous bushcricket studies (19), there are no signs that remov-ing the cuticle adversely affected auditory sensitivity. The population datain are combined from animals with an intact cuticle and animals withthe cuticle removed. Tympanum measurements were obtained by placingthe anterior tympanum perpendicular to the OCT beam. All measure-ments were acquired in vivo. In total, tympanum measurements wereacquired from 11 legs belonging to different animals. At the end ofthe experiment, the animal was anesthetized and subsequently killed bydecapitation.

Acoustic Stimulation. Broadband multitone “zwuis” complexes (43) wereused, following an open-field stimulation paradigm, using a ScanSpeakerR2904/700 005 loudspeaker placed 35 cm from the animal inside a sound-proof booth. The ipsilateral acoustic trachea opening on the thorax wasfacing the loudspeaker. Calibration of the sound pressure was done by tem-porarily replacing the animal holder by a microphone. The surfaces of theOCT probe, animal holder, etc., were covered with cotton wool in order to

minimize acoustic reflections. The most commonly used broadband stim-ulus consisted of 51 spectral components, spanning from 0.4 to 50 kHz(average frequency spacing: 990 Hz), presented at 75, 79, or 85 dB percomponent. All the spectral components had equal amplitudes, with lev-els expressed in decibels re 20 µPa (i.e., decibels SPL), but the stimulus phasewas randomized across frequency. Each stimulus was presented for 12 s, andinterstimulus intervals lasted for ∼60 s.

OCT Imaging and Vibrometry. Vibration measurements were obtained usingOCT. More details on the experimental setup are in ref. 25. OCT is an imag-ing technique, based on low-coherence interferometry. A spectral domain -optical coherence tomography system (Thorlabs Telesto TEL320C1) was usedfor interferometric imaging and vibration measurements. Its field of viewis 10 × 10 mm; its depth range is 3.5 mm. The working distance of theobjective was 24 mm. Cross-sectional (B scan) and axial images (A and Mscans) were triggered externally using monophasic rectangular pulses phaselocked to the acoustic stimulation system (Tucker Davies Technologies Sys-tem III) at a sampling rate of 111.6 kHz. The optics of our recording system(Mitotoyu IR imaging lens with a numerical aperture of 0.055) introduced anaxial point spread function of 6-µm full width half maximum and a lateralresolution (in the xy plane) of 13 µm (all assessed in air, with a refractiveindex of one). For our dorsal view measurements, the OCT measurementbeam was manually aligned with the dorsal–ventral anatomical axis of theCA. The vibration measurements that we made should therefore be favoringmeasuring vibrations along this axis.

The refractive index n of the various anatomical structures affects therecorded vibration magnitude. Phase data are not affected by n, whereasmagnitude is shifted upward by 20 log n≈ 3 dB for most fluids. The effectof correcting the data for n is illustrated in SI Appendix, Fig. S4, includingthe case where DW and CC have different n, motivated by the suggestionthat fluid composition between the CC and the DW may differ from that ofthe hemolymph canal around the CA (14, 17). Since the corrections are smalland uniform and the proper n values are unknown, we did not apply suchcorrections to the figures.

The anatomy of the distal end of the CA is quite distinct, and it is pos-sible to define the same anatomical structure across experiments. In orderto compare recorded positions across animals, we therefore used the dis-tal end of the hearing organ as an anchor point. The recorded structureswere labeled by the horizontal distance between the measuring beam andthe vertical line that intersects the distal end of the CA (the red line inFig. 1C). The depth resolution of the OCT allowed us to record the DWand the CC simultaneously, along one recording beam, at different posi-tions of the CA. That way, a DW measurement and a CC measurementobtained from the same beam shared the same distance from the distalend of the CA.

Response Analysis. Mechanical vibrations were obtained from 1) the DWand the CCs and 2) both tympana and septum. Responses were analyzedby Fourier transformation of the vibration waveforms derived from con-tiguous groups of three pixels in each M scan, where each pixel covers adepth of 3 µm, depending on the exact value of the refractive index ofthe structures. Each frequency component in a vibration measurement wasassessed using the Rayleigh test (p = 0.001) for significance of phase locking(44). Upon repeated recordings, vibration magnitude and phase are typicallyreproduced to within 0.5 dB and 0.02 cycle, respectively. The mechanicalmeasurements from the CA were normalized to vibrations recorded onthe distal end of the organ (green marker in Fig. 1C) analogous to nor-malization using the middle ear ossicles commonly used in the field ofcochlear mechanics (45). This accounted for imperfection in the calibrationof the acoustics. We made sure that the reference vibration measurementand the CA recordings were performed while using the exact same con-figuration, in particular the same height of the adjustable optical probe.The use of a mechanical reference rather than a microphone makes thedata less suited for the estimation of acoustic transfer functions, but itis adequate for the aims of the present study, namely the direct com-parison of vibrations across different locations in a single hearing organ.The absence of ripples in normalized magnitude–frequency data (e.g., Fig.3 D–F) indicates that any acoustic reflections had little impact on ourrecordings.

Different recorded positions are identified by their distance from themost distal part of the CA, as shown with a red vertical line in Fig. 1C.Data were analyzed using custom software developed in the MatLab (Math-Works, USA) environment. The spatial and spectral peaks were detected byusing a polynomial fit on a 15-dB range around the peak of the normalizedmagnitude data. The order of the polynomial varied from second to fourth,

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according to the number of fitting points that fulfilled the 15-dB-range cri-terion. In a minority of cases, manual exclusion of the fitting points wasapplied if the automated peak detection algorithm failed. Phase averaging(indicated as white stripes in Fig. 2B) was obtained as follows. The phases φk

of individual pixels in each subdomain (anterior and posterior tympanum)were determined by taking the angle of

∑k exp(iφk), where i is the square

root of −1.Group delay was estimated from the data in Fig. 3 M–O by fitting straight

lines to the portions of the phase-frequency curves that correspond to themagnitude peaks (highest 10 dB) in Fig. 3 J–L. The reported group delayestimates are the negative slopes of these fit lines. Wavelengths and phasevelocity were derived from the data in by fitting straight lines to the por-tions of the phase-position curves that correspond to the magnitude peaks(highest 10 dB) in Fig. 3 D–F. Wave number estimates (k in cycles per microm-

eter) are the negative slopes of these fit lines. Wavelength λ and phasevelocity c were derived from k by λ= 1/k and c = f/k, respectively, with fthe stimulus frequency.

Data Availability. Vibration measurement data have been deposited in Fig-share (https://figshare.com/authors/Marcel van der Heijden/5457377). Datafor the figures have been deposited in Figshare (https://figshare.com/articles/dataset/Vavakou Scherberich Nowotny vanderHeijden 2021/15292230).All other study data are included in the article and/or SI Appendix.

ACKNOWLEDGMENTS. We thank Ana-Maria Marchidan for help with theillustrations. This work was supported by Deutsche ForschungsgemeinschaftGrant 841/8-1.

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